Streptomycetes are Gram-positive, aerobic, filamentous soil bacteria, which belong to the order of actinomycetales. In an early stage of Streptomyces growth on a solid medium, spores germinate and subsequently develop into a vegetative mycelium of multinucleoid and branching hyphae with occasional cross-walls (Chater, 1993). After environmental signals such as nutrient depletion, aseptate aerial hyphae are formed, growing on the vegetative hyphae, the latter being used as a substrate. Eventually, the aerial hyphae form uninucleoid cells that develop into hydrophobic spores, which are budded off from the tips of the hyphae. One of the striking features of streptomycetes and other members of the order of actinomycetales is their ability to produce a wide variety of secondary metabolites, including many antibiotics, which are produced in temporal relation to the onset of morphological differentiation in surface-grown cultures (reviewed in Chater, 1989). The molecular processes regulating the events that lead to differentiation of Streptomyces are still largely unclear.
Most streptomycetes only sporulate on solid media, while growth in a liquid culture is restricted to the formation of a vegetative mycelium. This typically develops into an intricate network of hyphae, among others resulting in pellet formation, with only the most outwardly oriented sections showing high physiological activity, resulting in reduced growth rate and low yield of the desired product per unit of biomass. Furthermore, because of their filamentous morphology, high density fermentations of biotechnologically interesting streptomycetes often are highly viscous, resulting in a low biomass accumulation due to, for instance, aeration and mixing problems.
From this perspective, it is desirable that fragmentation of the mycelium in submerged cultures is stimulated, that branching of the mycelium is reduced, and that in general, the viscosity of the culture is reduced. The role and function of SsgA (SEQ ID NO:11) in the control of the morphology of actinomycetes have been disclosed in van Wezel et al. 2000 bcd and Kawamoto et al. 1997. However, so far no correlation has been made between the morphology of submerged cultures and enzyme or antibiotic formation in fermentations.
The choice of carbon source also has a major impact on morphological and physiological differentiation, as well as on morphology in liquid-grown cultures. In bacteria, the preferential use of readily metabolizable carbon sources is controlled via carbon catabolite repression (CCR), a mechanism well studied in Bacillus subtilis and in Escherichia coli. In both bacteria the phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a dominant role, controlling carbon utilization operons by specific (LacI, GalR) and pleiotropic regulators (CytR, CcpA). While Streptomyces coelicolor also has a PTS, its role is unclear (Parche et al., 1999; Butler et al., 1999).
Glucose kinase (Glk) is the key control point of CCR in S. coelicolor (Angell et al., 1992, 1994); glkA mutants are unable to grow on glucose and are deregulated in glucose repression of catabolite-controlled genes (Angell et al., 1992). Interestingly, introduction of the corresponding gene from Zymomonas mobilis resulted in restoration of glucose utilization, but failed to restore CCR (Angell et al., 1994). This suggests that CCR is mediated by glucose kinase through a regulatory site that is different from the catalytic site. Interaction with the PTS seems unlikely, as the system failed to phosphorylate Glk in vitro (Mahr et al., 2000). Due to the lack of a DNA-binding motif, Glk has been proposed to interact with transcription factors, such as GylR and MalR, which are responsible for both specific and global catabolite control of the glycerol and maltose regulons, respectively (Hindle et al., 1994; van Wezel et al., 1997).
There is also a logical link between CCR and Streptomyces development; the main signal for the initiation of aerial hyphae formation is nutrient depletion, suggesting a strong link between carbon metabolism and Streptomyces development. Indeed, many streptomycetes show relatively poor sporulation when grown on glucose-containing solid media, as compared to when mannitol or maltose is used as sole carbon sources. Also, submerged sporulation by Streptomyces griseus is repressed by glucose, as are several genes involved in aerial mycelium formation (van Wezel, unpublished data).
Although it is clear that the morphology of filamentous microorganisms is important from a biotechnological perspective and although there are some indications and leads on how to influence the morphology of filamentous microorganism, there is in general a need for tools to influence the morphology in general and, more specifically, a clear need for alternative approaches for obtaining enhanced fragmentation of the mycelium, in preferably submerged cultures, that branching of the mycelium is reduced and that the viscosity of high-density fermentation broths is reduced.